Method for treating plants

ABSTRACT

The present invention provides a method for increasing productivity of a plant by spraying Pink Pigmented Facultative Methylotroph (PPFM) on a plant. The invention also relates to increasing productivity of a plant under stress by applying Pink Pigmented Facultative Methylotroph (PPFM) to a plant and subsequently applying an aqueous solution containing methanol to the plant.

CROSS-REFERENCE TO RELATED APPLICATIONS

1. This application is a continuation-in-part of U.S. application Ser.No. 08/686,659, filed Jul. 26, 1996.

BACKGROUND OF THE INVENTION

2. 1. Field of the Invention

3. The present invention provides a method for treating plants byspraying plants with Pink Pigmented Facultative Methylotroph (PPFM)either alone or in combination with methanol during growth.

4. 2. Discussion of the Background

5. Improvements in crop yields is a highly active area of research, andas a result, today's farms are much more productive than theircounterparts from a century ago. However, as the world's populationincreases with a concomitant decrease in farming resources, more andmore emphasis is being placed on enhancing crop yields. Farmers arepresently seeking ways to expand their yields while limiting the use ofdangerous fertilizers and pesticides. An avenue of research which hasdeveloped from the desire to avoid harmful chemical crop treatments isthe treatment of seeds or the soil with non-toxic crop augmenter priorto sowing.

6. Schroth et al, U.S. Pat. No. 4,849,008, describes enhancing root cropyields by treating plant seeds with a specific growth promotingbacterial strain of the genus Pseudomonas. The bacterial strains may beapplied with a liquid carrier or in a paste.

7. Williams, U.S. Pat. No. 5,106,648, refers to a method of preparingcoated seeds by slurrying seeds with a microorganism, which has abeneficial effect on plants which grow from these seeds, a carriermedium and an adhesive polymer. This method is supposed to maintainmicroorganisms viable for extended periods of time.

8. Mann, U.S. Pat. No. 4,061,488, addresses treatment of plant seedswith spores from Bacillus uniflagellatus to enhance plant growth. It issuspected that root growth triggers the germination of these spores.

9. Polacco et al, U.S. Pat. No. 5,268,171, describe a method of alteringthe metabolism of a plant includes the steps of genetically altering atleast one commensal bacterium of the plant to alter the level and natureof urease activity produced by the plant.

10. Holland et al, U.S. Pat. No. 5,512,069, which is incorporated hereinin its entirety, describes methods for increasing the germination ofseeds by coating or impregnating them with PPFMs. PPFMs were firstdescribed in connection with plants more than 20 years ago when it wasdemonstrated that cell cultures of the leafy liverwort Scapania areroutinely associated with PPFMs (Basile et al, 1985, The Bryologist88(2):77). Since then these bacteria have been shown to be universallyassociated with plants, but the exact nature of the relationships hasremained obscure.

11. Nonomura and Benson, Proc. Nat'l. Acad. Sci. U.S.A., 89, 9794 (1992)teach that foliar sprays of aqueous (10-50%) methanol increased growthand yield of C3 crop plants in arid environments.

12. Nishio et al, in Proceedings of Twentieth Annual Meeting PlantGrowth Regulator Society of America, Ferguson, ed., pp. 8-13, 1993,teach that a 30% increase in dry matter and accelerated rate ofdevelopment of soybean plants can be achieved with a foliar-applied 15%methanol with fertilizer on a daily rotated basis.

13. Moore et al, U.S. Pat. No. 4,297,130 teach a method for the foliarfeeding of leguminous plants with a nonburning nitrogenous plant foodwhich is applied to the foliage of he plant at the R1-R4 floweringstage.

14. Joshi, U.S. Pat. No. 5,532,204, which is incorporated by referenceherein in its entirety, teaches a method of fertilizing leguminousplants for increase yield using methanol and urea based nitrogenfertilizer.

15. Despite these teachings, the use of methanol has not become routinein commercial application. Two reasons for this may be that (1) thetreatment is not universally effective under all cultural conditions oron all crops; and (2) the mechanism by which methanol works to affectyield has not been demonstrated convincingly.

16. Because methanol applications seemed to be most effective under highlight conditions and in C3 plants, it was suggested that methanol worksby inhibiting photorespiration (Nonomura et al, 1992, Proc. Natl. Acad.Sci. USA 89:9794-9798). This idea was supported by the observations thatthe treatment was enhanced by the addition of glycine to the spraymixture and that C4 plants (in which photorespiration is already low)did not respond. Fall and Benson (1996, Trends in Plant Sci. 1: 296-301)have elaborated on this explanation, suggesting possible alterations inmetabolism that would result in a greater than normal return ofphotorespiratory carbon to the chloroplast. However, further studieshave complicated the picture, and thus the mechanism by which methanolaffects plant growth is not clear.

17. Moreover, despite the above knowledge, producers of crop plantsreport “hit or miss” success with the above methods. Accordingly, amethod which provides reproducible increases in growth and yield isdesirable.

SUMMARY OF THE INVENTION

18. Accordingly, one object of this invention is to provide a novelmethod for treating plants which will result in increased yield and/orgrowth.

19. One object of the invention is to provide a method for increasingplant productivity (growth and yield) by applying a PPFM to a plant.

20. Another object of the invention is to provide a method forincreasing plant productivity (growth and yield) by applying a PPFM to aplant, followed by applying an aqueous solution of methanol and nitrogento the plant.

21. Yet another object of the invention is to provide a method forincreasing the productivity (growth and yield) of a plant under stress,followed by applying an aqueous solution of methanol and nitrogen to theplant.

22. With the foregoing and other objects, advantages and features of theinvention that will become hereinafter apparent, the nature of theinvention may be more clearly understood by reference to the followingdetailed description of the preferred embodiments of the invention andto the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

23.FIG. 1 shows a possible model for how PPFM bacteria mediate theeffect of methanol on plants.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

24. PPFMs have been reported from virtually all land plants examined.Corpe, J. Microbiol. Methods, 3:215-221 (1985). Members of all plantphyla contain associated PPFMs. While other-non-pink methylotrophs arealso associated with plants, PPFMs are the most persistent and are thepredominant phylloplane methylotrophs. PPFMs have the followingcharacteristics: (1) they are facultative methylotrophs; (2) they aredistributed ubiquitously on plants; (3) they are present in largenumbers; (4) they stimulate plant growth in vitro; (5) they participatein plant nitrogen metabolism; (6) they enhance seed germination; (7)they stimulate root growth; and (8) they manufacture cytokinins.

25. Suitable plants include any plant such as field crops, floweringplants (leguminous plants especially), conifers (pine trees), etc.Preferred leguminous plants include peanut, bean, pea and soybean.

26. The inventors have found PPFMs in all soybean tissues, includingseed and callus. The inventors classify PPFMs as Methylobacterium spp.,not only by their methylotrophy and pink pigmentation, but also due torestriction analysis of PCR-amplified 16S rRNA genes. Tsuji et al, J.Gen. Microbiol. 136:1-10 (1990). PPFMs isolated from soybean mostclosely resemble M. mesophilicum while isolates from other plants groupinclude such members as M. organophilum and M. extorquens. Thus, theterm PPFM as used herein is intended to encompass all pink pigmentedfacultative methylotroph bacteria. It should be understood that bothnaturally occurring, mutagenized and recombinantly obtained bacterialcan be used.

27. In a preferred embodiment, the PPFM is at least one PPFM selectedfrom the group consisting of M. mesophilicum, M. organophilum and M.extorquens. In a more preferred embodiment, M. mesophilicum is used.

28. PPFMs can be applied onto plant foliage, onto plant seeds or ontothe soil in which plants will be propagated.

29. In a first embodiment, PPFMs are applied to a plant at least onceduring the R1-R8 of growth. Critical stages of growth of plants(soybeans) are described by Fehr et al, “Stages of Soybean Development”,Iowa Sate University Press: Ames, Iowa, 1977. Preferably, viable PPFMsshould be present in an amount effective to increase the productivity(either yield or growth) of the plant by at least 1% compared with anuntreated plants. More preferably, viable PPFMs should be present in anamount effective to increase the productivity of the plant by at least2% compared with an untreated plant. Most preferably, viable PPFMsshould be present in an amount effective to increase the productivity ofthe plant by at least 5% compared with an untreated plant. Preferably,bacterial concentration of 10⁵ to 10¹⁰ bacterial cells/ml is considereda desirable range, although optimum values for specific plants andbacteria may be empirically determined.

30. In a second embodiment, PPFMs can be applied to plant seeds. Theapplication of PPFMs to the seeds of plants can consist of coating seedsusing known coating procedures such as those described in U.S. Pat. No.5,106,648, the contents of which are incorporated herein by reference.For example, seeds may be coated by slurrying seeds with the a solutionof PPFMs and air drying the resulting product, preferably at atemperature not greater than 30° C.

31. The proportion of composition to seed may be selected from the rangeof 0.1 to 25% by weight of the seed, preferably, 0.5 to 5% by weight andmost preferably 0.5 to 2.5% by weight, depending on the type of seed.

32. Alternatively, seeds can be immersed in a solution of PPFM such thata portion of the solution enters the seeds. Thereafter, the seeds can beplanted or dried for later planting. Alternatively, PPFMs can bedelivered to the seeds by vacuum infiltration or under pressure.

33. Viable PPFMs should be present in the coated seeds in an amounteffective to increase the germinability of a seed lot (e.g., 50 seeds)by at least 0.5% compared with an uncoated seed lot. Preferably, viablePPFMs should be present in an amount effective to increase thegerminability of a seed lot by at least 1% compared with an uncoatedseed lot. More preferably, viable PPFMs should be present in an amounteffective to increase the germinability of a seed lot by at least 2%compared with an uncoated seed lot. Most preferably, viable PPFMs shouldbe present in an amount effective to increase the germinability of aseed lot by at least 5% compared with an uncoated seed lot. In preferredcoatings, bacterial concentration of 10⁵ to 10¹⁰ bacterial cells/ml isconsidered a desirable range, although optimum values for specific seedsand bacteria may be empirically determined.

34. Treated seeds are then planted and grown under normal conditions.Alternatively, PPFMs can be applied to plants at any stage of theirdevelopment. Conventional spraying techniques can be used. The PPFMs canbe administered prior to, after, or concurrently with the methanolapplication.

35. In a third embodiment, PPFMs are applied to the soil in which theseed will be sown. Seeds are then sown in the treated soil.

36. The application of methanol and nitrogen is conducted at least onceduring the R1-R8, preferably R3-R5 stage of growth. Critical stages ofgrowth of soy bean, a leguminous plant, are described by Fehr et al,“Stages of Soybean Development”, Iowa Sate University Press: Ames, Iowa,1977.

37. A suitable aqueous solution of methanol and nitrogen in accordancewith the present invention is described in U.S. Pat. 5,532,204.

38. The amount of methanol (M₁) in the aqueous solution is from 10-50v/v % based on the total volume of water. The amount of methanol appliedto the foliage is 50 to 150, preferably 62.5 to 125, L ha⁻¹. Otheralcohols can be substituted for methanol such as ethanol, propanol,butanol.

39. The amount of nitrogen (N1) in the aqueous solution is from 10-100,preferably 10-25, most preferred 12.5, Kg ha⁻¹. Suitable nitrogensources include urea such as contained in urea based fertilizers. Othernitrogen sources can be used.

40. The ratio of M₁:N₁ is preferably 2.5:1.

41. The solution may contain further nutrients such as potassium andphosphorous. Particularly suitable sources of these nutrients are suchas potassium phosphate and potassium polyphosphate. Phosphorous andpotassium are each ordinarily used in amounts less than about 5 w/v %,based on the volume of the solution.

42. The solution containing methanol and nitrogen is suitably sprayed onthe plant using conventional means.

43. Having now fully described the invention, it will be apparent to oneof ordinary skill in the art that many changes and modifications can bemade thereto without departing from the spirit or scope of the inventionas set forth herein.

EXAMPLES Example 1

44. Application of PPFMs to plants was performed in accordance with themethods detailed in U.S. Pat. No. 5,512,069. Application of methanol toplants was performed in accordance with U.S. Patent No. 5,532,204.

45. Soybeans were treated with dry heat before planting to lower thepopulation of PPFMs on them (see U.S. Pat. No. 5,512,069). Untreatedseeds served as controls.

46. Under non-stressed (freely watered) conditions, application ofmethanol does not affect plant dry weight (a measure of yield),regardless of whether PPFMs are present. % gain over Treatment Mean dryweight +/− SD (g) controls PPFMs present, 6.85 +/− 1.69 78 Methanolapplied PPFMs absent, 5.03 +/− 1.19 80 Methanol applied

47. Under stress (wilting) conditions, application of methanol improvesgrowth (increase in plant dry weight) only when PPFMs are present. %gain over Treatment Mean dry weight +/− SD (g) controls PPFMs present,4.64 +/− 0.72 65 Methanol applied PPFMs absent, 2.69 +/− 0.65  6Methanol applied

48. The data show that when plants are not under water stress, thepresence or absence of PPFM bacteria is not an important factor indetermining growth rate following the application of methanol. Cured anduncured plants gained 80% and 78% in dry weight during the course of theexperiment. When plants are water stressed, however, methanol iseffective in stimulating growth only when PPFMs are present. Curedplants under stress gained only 6% in dry weight while plants with PPFMsgained 65% dry weight. Thus, PPFMs and methanol exhibit a synergisticeffect on plants under stressed conditions.

Example 2

49. To test the effects of foliar applications of methanol on soybean(Glycine max (L.) Merr. cv. ‘Corsica’ (maturity group IV)), fieldexperiments were conducted over three years. In the first year, methanolat 0, 62.5 and 125 liters×ha⁻¹ was combined with urea nitrogen at 0,12.5 and 25 kg×ha⁻¹ and sprayed with distilled water in a total volumeof 500 liters×ha⁻¹ on test plants at the R5 stage of development. LatronB-1956 (Rohm and Haas) surfactant at 0.1% was included in alltreatments. Treatments were applied from a knapsack sprayer in arandomized complete block design with four replications. Test blocksmeasured 5m×1.52m (7.6m²). Plants were harvested by hand, threshed, andseed yield determined by weight. All seed weights are reported at 13%moisture content. In subsequent years, only the concentration ofmethanol (62.5 liters×ha⁻¹) and urea (12.5 kg×ha⁻¹) found to be mosteffective in the first year were used as methanol and nitrogentreatments in field experiments.

50. To test the effects of foliar applications of methanol and/ornitrogen on PPFM populations, the numbers of these bacteria on leavesfrom plants in field experiments of the second and third years wereassayed. For the assay, leaves from four plants in each of the fourreplications of each treatment were harvested one week after spraying.Leaf material was rinsed, then ground in distilled water in a mortar andpestle. Serial dilutions of the grindate were plated on ammonium mineralsalts (AMS) medium (Cote, R., ed. 1984, ATCC Media Handbook (AmericanType Culture Collection, Rockville Md.) solidified with 15 g per literBacto-agar (Difco) and containing 15 μg per ml cycloheximide to reducecontamination from fungi. Plates were incubated at room temperature for10 days before counting. Colony counts of PPFM bacteria were used tocalculate colony-forming units per gram fresh weight of plant tissue(cfu/gfw).

51. To test whether PPFMS applied to leaves can substitute for methanol,spray applications of the bacterial alone and in combination withmethanol were included in field experiments in the third year.Experiments were conducted as described above with the followingtreatments tested: distilled water (control), methanol plus urea,methanol plus PPFM bacteria (1×10¹⁵ cfu×ha⁻¹), and PPFM bacteria(1×10⁻¹⁵ cfu×ha⁻¹) alone. The bacteria used in these treatments wereisolated from soybean seed coat on plates of AMS medium. From a singlebacterial colony, a liquid culture in the same medium was establishedand used to inoculate 12 liter batches of the bacteria in a Microfermfermentor (New Brunswick Scientific). Cells were harvested bycentrifugation and resuspended in distilled water for application toplants in the field.

52. To test whether the presence of PPFM bacteria is required for foliarapplications of methanol to be effective, greenhouse experiments wereconducted. Seeds of soybean were cured of their PPFM bacteria with dryheat as previously described (Holland et al, 1992, Plant Physiol98:942-948)). Cured and untreated seeds wee then plated in thegreenhouse in a soil-less potting medium (Pro-Mix BX), PremierHorticulture Inc.). During germination, and while the seedlingsestablished themselves, all plants were watered freely. After 4 weeks,both cured and untreated plants were divided into two groups (each groupconsisting of approximately 10 pots of 5 plants). One group of curedseedlings and one group of untreated seedlings were maintained as beforeunder freely-watered conditions. The remaining plants were wateredsparingly—and only when the plants showed signs of wilting. After 2weeks, half of the plants in each test group were harvested and dried toconstant weight (a zero-time control). The remaining plants were sprayedwith methanol (0.5 ml per plant) from an atomizer and were allowed tocontinue growing under their respective watering regimes for anadditional 2 weeks. At the end of this period, all remaining plants wereharvested and dried to constant weight.

53. As shown in Table 1 below, data from the first year show thatmethanol produced statistically significant increased in yield whenapplied at a rate of 62.5 liters×ha⁻¹, except in combination with highnitrogen (urea nitrogen at 25 kg×ha⁻¹). Methanol at a rate of 125liters×ha⁻¹ was effective in combination with urea at 12.5 kg×ha⁻¹.Highest yields were obtained from plants treated with methanol (62.5liters×ha⁻¹) and urea (12.5 kg×ha⁻¹). This combination was used in allsubsequent field experiments. TABLE 1 Treatment M₀ M₁ M₂ N₀ 2.00 2.59*2.28 N₁ 2.18 2.94* 2.57* N₂ 2.15 2.36 2.32

54. To assess the effect of methanol and urea on PPFM populations,counts of the bacteria were made from leaves following treatment. Asillustrated in Table 2 below, treatments of urea, methanol, and thecombination all resulted in statistically significant increases in thenumbers of PPFMs on leaves. Leaf PPFM populations were also examined bya leaf print method (Corpe, 1985, J. Microbiol. Methods, 3:215-221) thatallowed assessment of the relative abundance of the bacteria on leaftops vs. bottoms. These prints showed that the increase in PPFM numbersfollowing spray treatment was primarily due to increases of the bacteriaon the tops of leaves—the surfaces to which the treatment was applied.Yields from treated plants were also measured (Table 2) and showed thesame pattern of increase achieved in the first year experiments. Moresignificantly, the increases in yield correlate with the increases inPPFM numbers (R²=0.88). TABLE 2 Treatment M₀ M₁ N₀ 2.00 2.4* [0.1 × 10⁵][1.5 × 10⁵] N₁ 2.4 ? [1.6 × 10⁵] [1.8 × 10⁵]

55. Field experiments in the third year tested whether PPFM bacteriaapplied directly to the leaves of soybean could substitute forapplications of methanol. Table 3 summarizes the data from theseexperiments. When PPFMs were applied either alone or in combination withmethanol, yield increases of 70% over controls resulted. Treatments thatincluded the bacteria were more effective than treatment with methanoland urea. TABLE 3 Treatment M₁N₁ M₁, M₀N₀ 2.00 PPFMs PPFMs Yield 1.351.73 2.35 2.34 Mean = 1.94 LSD_(0.05)) = 0.17 cfu/gfw 0.7 × 1.3 × 1 ×1.7 × Mean = 1.2 × 10⁵ PPFMs 10⁵ 10⁵ 10⁵ 10⁵ LSD_(0.05)) = 0.2 × 10⁵

56. Although not intending to be bound by theory, the abovedescribeddata suggests that methanol works by stimulating the growth of PPFMsresident on the plant. Not only does the increase in their numbers thatfollows application of methanol correlate with yield increases, but inthe absence of the bacteria methanol does not stimulate growth.Furthermore, increasing the numbers of PPFMs on leaf surfaces byapplying them directly produces the same effect on yield as applyingmethanol.

57. These results support a model that explains how PPFM bacteriamediate the effect of methanol on plants as follows (see FIG. 1):

58. (1) Foliar application of methanol stimulates the growth of PPFMbacteria resident on plant leaves. Under high light and low waterpotential, neither plant nor bacteria are growing. Applied methanol isperceived by the bacteria as a sign that the plant is growing. Inresponse to this signal, the bacteria themselves begin to grow.

59. (2) Growing PPFM bacteria produce cytokinins (Freyermuth et al,1996, in Microbial Growth on Cl Compounds, eds. Lidstrom, M. E. andTabita, F. R. (Kluwer, Dordrecht), pp. 277-284).

60. (3) The cytokinin signal is perceived by the plant as signal forgrowth (Holland, 1997, Plant Physiol, 115:865-868).

61. (4) The plant grows, producing additional methanol as a wasteproduct (Obendorf, 1990, J. Exp. Bot. 41:489-495).

62. (5) PPFM bacteria are stimulated to continue to grow by the methanolproduced by the plant.

63. (6) The cycle continues.

64. This proposed mechanism suggests that applied methanol isresponsible for initiating a cycle of cross-talk between bacteria andplant that results in enhanced growth and yield even under poor growingconditions.

What is claimed as new and is desired to be secured by Letters Patent ofthe United States is:
 1. A method of increasing productivity of plantsunder stress which comprises the steps of: applying to a seed or foliageof a plant Pink Pigmented Facultative Methylotroph (PPFMs), and applyingan aqueous solution comprising an alcohol and nitrogen to foliage ofsaid plant.
 2. The method of claim 1 , wherein said alcohol is methanol.3. The method of claim 1 , wherein said plant is a bean, pea, soybean orpeanut plant.
 4. The method of claim 1 , wherein said PPFM is selectedfrom the group consisting of M. mesophilicum, M. organophilum, M.extorquens and mixtures thereof.
 5. The method of claim 4 , wherein saidPPFM is M. mesophilicum.
 6. The method of claim 2 , wherein said aqueoussolution comprises 1-50 v/v% methanol.
 7. The method of claim 1 ,wherein said nitrogen source is urea.
 8. The method of claim 2 , whereinthe ratio of methanol to nitrogen is 2.5:1.
 9. The method of claim 2 ,wherein the amount of methanol applied to said plant is from 50 to 150 Lha⁻¹.
 10. The method of claim 1 , wherein the amount of nitrogen appliedto said plant is from 10-100 Kg ha⁻¹.
 11. The method of claim 1 ,wherein said PPFMs are applied to seeds of said plant and thereaftersaid aqueous alcoholic solution is applied to plants propagated fromsaid seeds.
 12. A method of increasing productivity of plants whichcomprises the steps of: applying to the foliage of a plant PinkPigmented Facultative Methylotroph (PPFMs).
 13. The method of claim 13 ,wherein said plant is a bean, pea, soybean or peanut plant.
 14. Themethod of claim 13 , wherein said PPFM is selected from the groupconsisting of M. mesophilicum, M. organophilum, M. extorquens andmixtures thereof.
 15. The method of claim 15 , wherein said PPFM is M.mesophilicum.